Fig. 3

DUX4c is immunodetected in myofibers either hypotrophic or with an unusual shape. DUX4c, desmin, and laminin-α2 were detected using the rabbit anti-DUX4c serum, mouse anti-desmin and rat anti-laminin-α2 sera followed by appropriate secondary antibodies coupled to AlexaFluor 488 (green), 555 (red), or 647 (purple), respectively. Nuclei were stained with DAPI (blue). Staining was observed by epifluorescence microscopy. A DUX4c immunostaining in hypotrophic fibers with either a rectangular (box 1) or a flat (box 2) morphology next to other myofibers with either peripheral or central (#) nuclei. DUX4c was also observed as short lines at the periphery of these adjacent fibers. Magnified box 1 shows DUX4c detection around the whole myofiber periphery, around one peripheral nucleus and next to a central nucleus (#). We also observed next to the peripheral nuclei, a DUX4c staining in the adjacent fibers (arrowhead) and a partial co-detection with desmin (arrow). Magnified box 2 shows DUX4c staining in dots, one of which is inside the myofiber and co-localizes with desmin at its two tips (arrows), and DUX4c again appears as a line in an adjacent fiber (arrowhead). As previously published [12], desmin is also detected without DUX4c staining. B DUX4c detection as in (A, box 2) (box 1) and in a normal-size fiber presenting an unusual shape (box 2) next to adjacent fibers with central nuclei (#). Magnified box 1 shows a very flat myofiber. DUX4c is detected around the two close nuclei and at the fiber tips although intense desmin is present. Next to this fiber, another nucleus (§) presents an intense desmin staining, mainly on one side, co-detected with an intense laminin-α2 signal. Magnified box 2 shows the myofiber with an abnormal shape at one round tip presenting cytoplasmic DUX4c (arrows) either at this tip or next to a large peripheral nucleus. The close adjacent fibers also present cytoplasmic DUX4c either at one tip or next to a large peripheral nucleus (arrowheads). C Another myofiber showing unusual shape with two triangular tips containing DUX4c labeling (white arrows). One tip also presents an intense internal desmin staining that is co-detected with intense laminin-α2 signal (yellow arrows)